Basal-like breast cancer (BLBC, or triple-negative) is a highly lethal form of the disease for which effective therapeutics are lacking. This lethality is though to be due in part to the increased numbers of tumor- initiating (or stem) cells measured in BLBCs. The epidermal growth factor receptor (EGFR) is commonly expressed in these tumors, but inhibitors against the receptor have shown measurable but limited activity in breast cancer clinical trials. In contrast, EGFR is an effective target in multiple other solid tumor types. Even though clinical trials using EGFR inhibitors continue in breast cancer, the lack of efficacy of these agents in this disease remains poorly understood. BLBCs also show frequent hyperactivation of Notch receptors, which are oncogenic in the mammary gland. The four family members are commonly co-expressed in breast cancer cells where they can play opposing or redundant roles. Their activation is dependent on the enzymatic activity of the gamma secretase complex, inhibitors of which (GSIs) are being trialed in the clinic. These drugs globally inhibit all four receptors and can produce significant toxicity, motivating investigation into less toxic approaches to blocking Notch signaling. Fortunately, many Notch-dependent processes are sensitive to gene dosage and are receptor specific. Therefore, complete and pan-receptor inhibition may not be necessary for all therapeutic applications involving Notch receptors. We have recently reported a synthetic lethal relationship between the Notch and EGFR pathways (Dong, et al., Cancer Research, July 2010). Through the following aims, we will test the hypothesis that Notch and EGFR receptors function as cooperating oncoproteins required for BLBC cell survival. Specific Aim 1. To examine the effect of combined Notch-EGFR pathway inhibition on tumor-initiating cell survival in BLBC tumors. We will assess the relative ability of combined pathway inhibition to eradicate the tumor-initiating and more differentiated fractions of breast cancer cells. We will also begin to determine the efficacy of combined pathway inhibition against primary human BLBC tumors using a novel technology wherein these cancers are grafted into immunodeficient mice, and investigate the requirement for functional Notch- EGFR pathways in a broader panel of BLBC cell lines that show variable EGFR expression. Specific Aim 2. To determine the ability of partial Notch pathway inhibition to induce synthetic lethality when combined with EGFR blockade in BLBC cells. Partial pathway inhibition will be achieved through two strategies, dose modifications of GSI and receptor-specific knockdowns. In combination with EGFR blockade, we will examine the extent to which these approaches induce cell death and how they affect the viability of tumor-initiating and differentiated cells. Conversely, through forced expression of activated receptors, the ability of individual receptors to suppress cell death and expand the tumor-initiating population in the presence of GSI and EGFR inhibitor treatment will be measured.